Jacob Kjell

Injury to the spinal cord results in chronic disruption of the spinal tracts and permanent loss of bodily functions. One reason that spinal cord circuits do not regenerate is the permanent scar at the injury-site and the dramatically altered surrounding extracellular environment that remains non-permissible to axon growth. Our current knowledge concerning the cellular and molecular composition of the scar and the roles of various cell-phenotypes primarily stem from experimental rodent models, while our knowledge associated to the human spinal scar remains limited. Many aspects of cell/phenotype composition, drug target expression, and composition of ECM and glial/fibrotic components remain unknown. Hence, the gap in our knowledge between that of spinal pathobiology in humans and rodents has grown exceedingly large which is information of potential great consequences to translation of treatments. In this project we aim to map the cellular and molecular composition and drug targets in the human spinal cord scar. We possess chronic spinal scar samples that stem from 7 patients, whose tissue was collected as part of a clinical trial. We will map the human spinal cord scar combining single nuclei RNA sequencing (snRNAseq) and in situ sequencing (ISS) for spatial gene expression analysis on a single cell level. Furthermore, we will use mass spectrometry for an unbiased and comprehensive proteome of different regions of the scar tissues. The methods permit us to identify various cell-types/phenotypes, a multitude of extracellular matrix proteins, and many drug targets, and new potential biomarkers. The identification of various scar regions for tissue extraction for the mass spectrometry will be supported by the ISS data. Furthermore, we will determine similarities and discrepancies to the chronic spinal cord scar in rodents, including bioinformatic comparisons to newly acquired single cell gene expression datasets of the rodent scar. We are right now starting the snRNAseq measurements and have already performed pilot and optimization for both ISS and mass spectrometry. Notably, we will also use uninjured human spinal cord samples as control tissues, and we intend to make the data from this analysis available in online. Moreover, we will confirm cellular and molecular features associated to various distinct research questions using immunohistochemistry and in situ the tissue sections. Towards the end of the 2-year grant period and beyond, we intend to investigate the functional roles of various candidate targets in vitro and in vivo. In summary, we believe this translational map of the spinal scar can ensure relevant information is used for the development of future repair strategies.